多光子荧光成像在生物医学研究中应用广泛。我们演示的1700nm波段三光子荧光成像充分发挥多光子成像的深度优势，对活体动物脑成像深度超过其他多光子成像技术。荧光标记物是多光子荧光成像的材料基础，其多光子发射谱是成像首要关注的光学参数之一。目前荧光标记物在该波段三光子发射谱未知，严重妨碍了：基本光化学原理——卡莎定则的验证；采集效率及成像深度的进一步优化；量子点这种特定标记物在深层脑区测温中的应用。为此，本项目拟开展1700nm波段荧光标记物三光子发射谱测量技术及应用研究。在发射谱测量系统建造的基础上，对多种标记物进行三光子发射谱测量，并与单光子发射谱对比，明确是否遵循卡莎定则。根据测量结果，优化系统采集效率、提升三光子成像深度，并研究量子点三光子发射谱在活体小鼠脑部测温中的应用。研究成果将为该波段三光子成像荧光标记物选择提供完备的发射谱数据库，并在活体动物深层成像、温度测量中体现应用价值。

Multiphoton fluorescence microscopy has found widespread applications in biomedical research. Our recently developed 3-photon fluorescence microscopy excited at the 1700-nm window fully exploits the deep-tissue penetration capability of multiphoton microscopy (MPM), and enables larger imaging depth in animal brain imaging in vivo compared with other MPM technologies. Fluorescent probes are the material basis for multiphoton fluorescence microscopy, whose multiphoton emission spectrum is one of the key optical parameters. Thus far, the 3-photon emission spectra for fluorescent probes excited at the 1700-nm window remain unknown. This severely hampers: verification of Kasha’s rule-the fundamental principle in photochemistry, optimization of detection system in the multiphoton microscope and the resultant imaging depth, and application of quantum dots to deep-brain temperature measurement. In order to tackle these problems, here we propose to do systematic research on measurement technology and application of 3-photon emission spectrum for fluorescent probes excited at the 1700-nm window. Based on emission spectrum measurement system development and building, we will measure and compare the 3-photon and 1-photon emission spectra of a variety of fluorescent probes, to test whether Kasha’s rule is still valid. Based on the measured results, we will also optimize the collection efficiency of the multiphoton microscope and the resultant imaging depth, and investigate the application of 3-photon emission spectrum of quantum dots to measuring the mouse brain temperature in vivo. Our results will establish a complete 3-photon emission spectrum database for fluorescent probes, which readily finds practical applications in deep-tissue imaging and in vivo temperature measurement.